There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate, quantitative determinations of biological substances, which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, seminal fluids and other biological fluids has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment. In addition, there is an interest in the attachment of biological macromolecules onto polymer particles. Such immobilized biomacromolecules have found applications in medical diagnostics, reaction catalysis, and in protein and deoxyribonucleic acid (DNA) microarrays.
One particular type of diagnostic method is an agglutination test, which is useful for the detection of antigens, which have multiple sites for antibody reactivity. In such a test, anti-body molecules can be bound in a suitable fashion to water-insoluble particles. Antibody-antigen reaction at multiple sites causes the particles to agglutinate and precipitate. Suitable separation and detection means have been devised to make the agglutinate readily observable, including for example, the use of particles containing a tracer material as described in U.S. Pat. No. 4,997,772 and in references cited therein.
Another useful method for detecting biological substances in fluids is what is known in the art as a “sandwich” assay. Such an assay involves “sandwiching” the compound of interest (such as an antigen) with two or more receptor molecules (such as antibodies) which complex with the compound at different and noninterfering sites. Examples of such assays arc described, for example, in U.S. Pat. No. 4,486,530. In most sandwich assays, one or more of the receptor molecules are suitably immobilized on an insoluble carrier such as small particles, membranes, plates, test wells or similar objects.
Attachment of antibodies or receptor molecules to insoluble carrier materials has been achieved in the past in a number of ways. Early work relied on adsorption of the molecules, but it was realized that adsorption is generally not a strong method of attachment. Later researchers found that the molecules could be covalently attached by reaction of certain functional groups of the molecules with specially designed reactive groups on the carrier material. For example, proteins have been attached by reacting carboxy groups of particles or supports with an activating compound, which renders the groups reactive with amino groups of a protein. Carbodiimides are examples of useful activating compounds.
Immunological compounds may be immobilized on polymer particles. Although immunological compounds may be directly attached to the particles, direct attachment may deactivate the immunological compounds. Yet, it is desired to strongly attach those compounds to the particles.
Various reagents have been prepared with particles having reactive groups such as epoxides, aldehydes, amino groups and diazonium salts. All of these groups have disadvantages. Epoxide groups are not stable, so that the particles cannot be stored for very long. Particles having aldehyde groups generally tend to agglutinate prematurely. The aldehyde groups also prematurely oxidize, thereby losing binding activity. Particles with amine groups, like carboxylated materials, require an additional activation step for attachment. Diazonium compounds are unstable and therefore undesirable to work with. EP 320715 and U.S. Pat. No. 5,374,516 disclose latex particles, which contain repetitive vinylsulfonyl units. These units, however, are bound directly to the particle surface. Furthermore, they are of a size range, which is too small for use in assays, which require optical imaging.
U.S. Pat. No. 4,582,810 and PCT Publication 84/03358 describe the attachment of avidin to latex particles having free carboxyl groups on their surfaces. As described therein, the conventional procedure for covalently attaching avidin to the particles involves the use of a water-soluble carbodiimide in an activation step. While producing reagents, this procedure tends to activate the exposed reactive groups of the protein avidin as well as the carboxyl groups on the particles. The result is intramolecular and intermolecular crosslinking or polymerization of avidin, and a significant portion of the reagent is impaired from complexation with biotin. In addition, there may be premature agglutination of the insolubilized reagent due to the cross-reactivity of the activating compound. These problems present a serious economic loss as well as an impairment of diagnostic sensitivity. It has also been evident that carbodiimides provide a reactive intermediate for avidin attachment, which is unstable and must be used immediately. The reactive units of the carboxylic acid particles are bound directly to the particle surface, which can limit the accessibility of biomacromolecules immobilized therein.
Macromolecular Rapid Communications Vol. 15 p. 909–915 (1994) reports the immobilization of enzymes to soluble stabilizer polymer arms protruding from the surface of a polymer particle. Enhancements in accessibility of the enzyme to target substrates are observed over enzymes covalently bound directly to the particle surface. However, the enzyme was reversibly adsorbed to the stabilizer arms and was not covalently bound. In addition, the stabilizer arms contained only carboxylic acids as their reactive functionality, so if covalent attachment was desired, it would require the use of a coupling agent and a subsequent preparative step.
Hence, reagents, which are covalently attached to a water-insoluble particle, would be very useful in diagnostic methods. It would also be desirable to have reagents for immobilizing immunological species without directly attaching the species to the insoluble carrier material, while achieving an attachment that is stronger than that achieved through mere adsorption. It would also be advantageous if the immobilization could be afforded without the addition of further chemical reagents.